1. Field of the Invention
The present disclosure relates to an improved method of producing terminally differentiated neuronal cells such as dopaminergic and serotonergic neurons from pluripotent embryonic stem cells such as human embryonic stem cells. The dopaminergic and serotonergic neurons generated according to the present disclosure may serve as an excellent source for cell replacement therapy in neurodegenerative disorders and neuronal diseases.
2. Description of Related Art
Neurodegenerative disorders and neuronal diseases such as Parkinson's disease, Alzheimer's disease, and schizophrenia are destructive diseases that are becoming ever more prominent in our society. Many of these neurological disorders are associated with dopaminergic or serotonergic neurons. Dopaminergic neurons reside in the ventral and ventro-lateral aspects of the midbrain, and control postural reflexes, movement, and reward-associated behaviors. These neurons innervate multiple structures in the forebrain, and their degeneration or abnormal function is associated with Parkinson's disease, schizophrenia, and drug addiction (Hynes et al., 1995, Cell 80:95-101). Serotonergic neurons are concentrated in the ventral and ventro-lateral aspects of the hindbrain and innervate most parts of the central nervous system including the cerebral cortex, limbic system and spinal cord. These neurons control levels of awareness, arousal, behavioral traits, and food intake, and their abnormal function has been linked to aggression, depression, and schizophrenia (Jacobs and Gelperin, 1981, Serotonin Neurotransmission and Behavior. The MIT Press, Cambridge, Mass.). Serotonergic dysfunction may also play a role in the pathophysiology of various psychiatric, neurologic, and other diseases, for example, mental depression (Asberg et al., 1986, J. Clin. Psychiatry 47:23-35), suicide (Lester, 1995, Pharmocopsychiatry 28(2):45-50), and violent aggressive behavior (Brown et al., J. Clin. Psychiatry, 1990, 54:31-41; Eichelman, 1990, Annu. Rev. Med. 41:149-158).
Parkinson's disease is a progressive neurological disorder caused by the degeneration of nerve cells (neurons) in the region of the brain that controls movements. This degeneration creates a shortage of the brain signaling chemical (neurotransmitter) known as dopamine, causing the movement impairments that characterize the disease. Pathological studies indicate that loss of dopaminergic neurons in the substantia nigra contributes to Parkinson's disease. For example, bilateral lesions of the nigrostriatal pathway produce a syndrome in experimental animals that is quite similar to the observed motor dysfunctions observed in Parkinson's disease: resting tremor, rigidity, akinesia and postural abnormalities. Bilateral lesions of the nigrostriatal pathway caused by 6-hydroxydopamine (OHDA) caused profound akinesia, adipsia, aphagia and sensory neglect in rodents (Ungerstedt, 1971, U. Acta Physiol. Scand. Suppl. 367:95-121; Yirek and Sladek, 1990, Annu. Rev. Neurosci. 13:415-440).
In parkinsonism, changes in the status of dopaminergic receptors may be dependent on the stage of progression of the disease. The hallmark of parkinsonism is a severe reduction of dopamine in all components of the basal ganglia (Hornykiewicz, 1988, Mt. Sinai J. Med. 55:11-20). When dopamine is depleted, various other areas in the brain such as the thalamus, globus pallidus, and the subthalamic nucleus start to malfunction. Since these areas send signals to other parts of the brain, malfunctions in these small areas can lead to widespread brain dysfunction.
The prevalence of Parkinson's disease varies widely from 82 per 100,000 in Japan and 108 per 100,000 in UK, to nearly 1% (approximately 1 million) of the population in North America. In India, the prevalence rate of Parkinson's disease is 14 per 100,000 in North India, 27 per 100,000 in South India, 16 per 100,000 in East India, and 363 per 100,000 for the Parsi community in Western India. While Parkinson's disease is currently considered incurable, a variety of medications are available that provide symptomatic relief from Parkinson's disease, including Levodopa, Bromocriptine, pergolide, selegiline, anticholinergic, and amantadine. Although these drugs may provide relief from the symptoms of Parkinson's disease, they often have significant side effects. Moreover, these drugs neither cure the disease nor slow down the progressive loss of neurons, and only relieve the symptoms, with the beneficial effects often wearing off with time. Some patient become less responsive to medication, while others become hypersensitive and develop dyskinesias.
These unsatisfactory outcomes have led to development of other strategies for treating this disease, such as dopa-receptor agonist therapy and surgical approaches that include pallidotomy, deep brain stimulation (DBS) of the globus pallidus, and attempts to interrupt network abnormalities by destroying overactive brain areas or placing DBS electrodes to quiet these area. Although these and other types of surgery for patients with Parkinson's disease patients have produced some beneficial results, the long-term effects of such surgeries are not yet known. These treatments also have certain limitations and side effects.
Another strategy being pursued for this incurable disease is gene therapy. Discovery of the molecular basis of neurological disease and advances in gene transfer systems have allowed focal and global delivery of therapeutic genes for a wide variety of central nervous system disorders. But gene therapy has certain limitations such as stability and regulation of transgene expression, and safety of both vector and expressed transgenes (Costantini et al., 2000, Gene Therapy 7: 93-109). Vectors shown to be used for gene therapy include but are not limited to Herpes Simplex Virus type-1 (HSV-1) (During et al., 1994, Science 266:1399-1403), adeno-associated virus vector (AAV) (During et al., 1998, Gene Therapy 5:820-827), retrovirus, HSV/Epstein-Barr Virus (HSV/EBV) hybrid vector, and HSV/AAV hybrid vector. One gene therapy approach has been found useful to treat an animal model of Parkinson's disease. An encapsulated, genetically engineered cell line releasing the neuroprotective molecule, glial cell line-derived neurotrophic factor gene (GDNF), and a lentiviral vector encoding the GDNF gene improved graft survival and differentiation, thereby accelerating behavioral recovery in the animal model (Zurn et al., 2001, Brain Res Rev. 36:222-229; Date et al., 2001, Cell Transplant 10:397-401). Gene therapy using neural stem cells has also been found to be effective in expressing therapeutic levels of GDNF in vivo (Akerud et al., 2001. J. Neurosci. 21:8108-8118).
Cell implantation is another therapeutic strategy that offers the hope of replacing nerve cells lost in Parkinson's disease, as well as other neurodegenerative disorders and neuronal diseases. Clinical trials with fetal tissue transplantation, still underway, have developed methods for implanting cells into the brain and demonstrated the viability of this concept, as well as produced promising results for at least some patients. Attempts have also been made to transplant precursors of dopaminergic nerve cells directly into the striatum of patients with Parkinson's disease, and transplantation of human fetal or embryonic dopaminergic neurons have been found to have a beneficial effect on patient with Parkinson's disease (Freed et al., 2001, N. Engl. J. Med. 344:710-719). Data suggests, however, that anatomical repair of the pathway rather than ectopic placement of the graft may be required to obtain complete recovery (Winkler et al., 2000, Prog. Brain Res. 127:233-265). Additionally, fetal nigral transplant therapy requires human fetal tissues from at least 5-10 fetuses in order to have a clinically reliable improvement in the patient, which poses enormous ethical, legal, and safety issues. Thus, there is an urgent need for alternative sources of neuronal cells such as dopaminergic neurons to treat neurodegenerative disorders and neuronal diseases.
Recently, a renewable source of neural stem cells was discovered in the adult human brain. Neural stem cells with the capacity to renew themselves and form all cell types of the brain offer a potentially unlimited supply of dopamine producing brain cells, thus promising an entirely new therapeutic approach to neurodegenerative disorders and neuronal diseases (Eriksson et al., 1998, Nature Medicine 4:1313-1317). It has been reported that cultures of neural stem cells derived from the embryonic human forebrain can be expanded up to ten million fold in vitro. These adult neural stem cells have been transplanted into adult rats that are a well characterized model of Parkinson's disease. The cells in this animal model survived for up to a year after transplantation, differentiated into neurons, and were able to decrease motor disorders in some of the experimental animals (Svendsen et al., 1997, Exp. Neurol. 148:135-146). Unfortunately, adult neural stem cells have a limited life span in tissue culture (Kukekov et al., 1999, Exp. Neurol. 156:333-344).
One viable alternative source of dopamerinergic neurons, and other neurons that may be used to treat various neurodegenerative disorders and neuronal diseases, are pluripotent embryonic stem (ES) cells, in particular human ES cells. ES cells can proliferate indefinitely in an undifferentiated state and are pluripotent, which means they are capable of differentiating into nearly all cell types present in the body. Because ES cells are capable of becoming almost all of the specialized cells of the body, they have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage, and the like. ES cells can be derived from the inner cell mass (ICM) of a blastocyst, which is a stage of embryo development that occurs prior to implantation. Human ES cells may be derived from a human blastocyst at an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. ES cells derived from the ICM can be cultured in vitro and under the appropriate conditions proliferate indefinitely.
ES cell lines have been successfully established for a number of species, including mouse (Evans et al., 1981, Nature 292:154-156), rat (Iannaccone et al., 1994, Dev. Biol., 163:288-292), porcine (Evans et al., 1990, Theriogenology 33:125-128; Notarianni et al., 1990, J. Reprod. Fertil. Suppl. 41:51-6), sheep and goat (Meinecke-Tillmann and Meinecke, 1996, J. Animal Breeding and Genetics 113:413-426; Notarianni et al., 1991, J. Reprod. Fertil. Suppl. 43:255-60), rabbit (Giles et al., 1993, Mol. Reprod. Dev. 36:130-138; Graves et al., 1993, Mol. Reprod. Dev. 36:424-433), mink (Sukoyan et al., Mol. Reprod. Dev. 1992, 33:418-431), hamster (Doetschman et al., 1988, Dev. Biol. 127:224-227), domestic fowl (Pain et al., 1996, Development 122(8):2339-48), primate (U.S. Pat. No. 5,843,780), and human (Thomson et al., 1998, Science 282:1145-1147; Reubinoff et al., 2000, Nature Biotech. 18:399-403). Like other mammalian ES cells, human ES cells differentiate and form tissues of all three germ layers when injected into immunodeficient mice, proving their pluripotency. Published reports show that human ES cells have been maintained in culture for more than a year during which time they retained their pluripotency, self-renewing capacity, and normal karyotype (Thomson et.al., 1995, PNAS 92:7844-7848).
Studies have shown that ES cells can be differentiated into neural progenitor cells (Zhang et al., 2001, Nature Biotech. 19:1129-33; WO 01/88104; U.S. Ser. Nos. 09/872,183, 09/888,309, 10/157,288; WO 03/000868; each specifically incorporated herein by reference). These cells can then be further differentiated into dopaminergic neurons (Rolletschek et al., 2001, Mech. Dev. 105:93-104). An initial step in the differentiation of ES cells can be the formation of embryoid bodies, for example 1 μM of retinoic acid promotes neural differentiation into embryoid bodies (Bain et al., 1995, Dev. Biol. 168:342-357). While retinoic acid can be used to generate neural cells, retinoic acid is a strong teratogen. Several reports have been published on the differentiation of ES cells into dopaminergic neurons by using stromal cell inducing activity (SIDA) (Kawasaki et al., 2000, Neuron 28:1-20), by expressing nuclear receptor related −1 gene (Nurr-1) (Kim et al., 2002, Nature 418:50-56), or by transplanting undifferentiated ES cells directly into the mouse model (Bjorklund et al., 2002, Proc. Natl Acad. Sci. 99:2344-2349). Lee et al. (2000, Nat. Biotechnol. 18:675-79) reported a method for differentiating ES cells into neural progenitor cells and into dopaminergic and serotonergic neurons in vitro. All of these experiments, however, were carried out using mouse ES cells, and the differentiation protocols yielded dopaminergic neurons ranging from 5-50%. About 20% of the mouse ES cells developed into dopaminergic neurons in the study by Lee et al. (WO 01/83715) and 5-50% in the study by Studer et aL. (WO 02/086073). While dopaminergic neurons have also been differentiated from human ES cells, yields of only 5-7% of dopaminergic neurons, as a percentage of total cells in the population, have been obtained (WO 03/000868).
Parkinson's disease is thought to be a particularly suitable clinical target for a cell transplant strategy since it is characterized by the selective and gradual loss of dopaminergic neurons in the substantia nigra of the midbrain. The loss of dopamine-producing neurons within this specific brain site leads to abnormal firing of nerve cells that results in patients being unable to control or direct their movements. But large numbers of dopaminergic neurons are required for cell replacement therapy. Therefore, alternate protocols are needed for deriving dopaminergic neurons from human ES cells more efficiently, which will both accelerate the availability of this treatment for Parkinson's disease and increase the success rate of treatment. Additionally, these dopaminergic neurons can be utilized in vitro to help identify substances that will prevent or reduce death of dopamine producing brain cells in neurodegenerative disorders and neuronal diseases.